Advancing Enantiomer Analysis

The Problem

The measurement of chirality and enantiomeric excess (ee) of multi-component mixtures is of profound importance in pharmaceuticals, food, fragrance and agrochemicals. Normal mass spectrometry (MS) is chirally blind and prior enantiomeric separation (chiral GC, LC) or enantiomer selective chiral complexation is needed before MS detection. Can we eliminate these often quite elaborate prior chiral preparation phases on enantiomers? Or, put another way, how can we detect the two enantiomers and measure their ee in multi-component chiral mixtures directly by MS?

Background

I spent some 35 years in academia. I was trained as an experimental physicist during my undergraduate and PhD studies at the Radboud University Nijmegen, the Netherlands. I moved further into the field of physical chemistry during my postdoc (around 1990) at the California Institute of Technology, Pasadena, and then moved to LaserLaB VU University Amsterdam, where I climbed the academic ranks (assistant, associate and full professor) until early 2015.

In 2008, we (my students, Arno Vredenborg and Wim Roeterdink, and our technician, Rob Kortekaas) had constructed a very exciting new lab in my group in Amsterdam to study the ‘intimate dance’ of electrons and nuclei in chemical bond breaking, using ultrafast lasers and advanced single particle coincidence imaging detectors. The research in our new coincidence lab was focused on advancing our fundamental knowledge of chemical photodynamics. And we also aimed to actively steer and control these photochemical reactions by playing with the colors and phases of our femtosecond laser. And thus we entered the young field of coherent control of chemical dynamics, where we wanted to manipulate the outcome of chemical bond breaking with ultrafast shaped laser fields. In fact, lasers have been a recurring theme in my research since my first experimental physics projects in the mid-80s.

At the International Conference on Stereodynamics of Chemical Reactions in Dalian, China, during the fall of 2008, I presented some first results obtained with our latest ‘toy’ in Amsterdam. On the last day of the conference, I was inspired by a mostly theoretical talk on chiral discrimination in molecular collisions. I had never worked on chiral molecules and after the talk I discussed chirality in molecules with my longtime collaborator, Peter Rakitzis (University of Crete, Greece). Peter pointed me to recent work by someone else in our community, Ivan Powis (University of Nottingham, UK). In 2000, Ivan had published the first quantitative calculations of the asymmetry in the angular distribution of photoelectrons that are ejected following the single photon ionization (using circular polarized light) of chiral molecules, such as alanine. Some 25 years earlier, it was predicted theoretically by Burke Ritchie (Argonne National Laboratories, USA) that when you photoionize a chiral molecule with a circular polarized photon, the angular scattering distribution of electrons would be asymmetric along the propagation direction of the photon. Ritchie used a model potential that was not directly related to a real molecule. However, he predicted large asymmetries (~10 percent) and later this ‘forward-backward asymmetry’ was coined photo electron circular dichroism (PECD) by Ivan. After that publication in 1976 not much happened – Ritchies’ prediction seemed almost forgotten until Ivan started some realistic calculations on real molecules in 2000.

In 2003, Ivan also started to measure this PECD effect experimentally in collaboration with a team headed by Laurent Nahon at the Soleil synchrotron facility near Paris. To photoionize a molecule you need an energetic photon (typically about 9-12 eV) and the Soleil synchrotron had a beam line where they could also control the polarization of that photon. In fact, they could switch the polarization of the beam of vacuum ultraviolet (VUV) photons from left circular polarized (LCP) to right circular polarized (RCP), and to linear polarized, all with good purity. Such a feat was far from trivial for synchrotrons around early 2000. Ivan and the Soleil team measured large asymmetries in the various chiral molecules they studied. The enantiomeric sensitivity of PECD that they observed in the electron angular distribution was typically around 1-10 percent, two to three orders of magnitude larger than the sensitivity of the conventional technique of absorption (vibrational) circular dichroism.

In another collaboration with Uwe Hergenhahn at the BESSY synchrotron in Berlin, Ivan demonstrated the same chiral phenomenon could be observed in X-ray core-level ionization. These exciting new developments certainly proved that PECD could be a very sensitive technique to detect molecular chirality; unfortunately, not many researchers have a synchrotron in their lab. Indeed, access to large-scale facilities that can provide the appropriate photon beam characteristics and the electron-imaging detector necessary is somewhat limited.

The Solution

During my stay in Dalian, I wondered what would happen with the photoelectron angular asymmetry, if you could use (for instance) three circular polarized photons (each with much less energy) to ionize a chiral molecule. I had ultrafast lasers in my Amsterdam lab and, because of my postdoc work at Caltech, I knew that it was very easy to ionize a molecule with such an intense laser through multi-photon ionization. With an intense pulsed laser, the molecule easily absorbs three 3 eV photons (~400 nm) and becomes ionized. The Monday after the Dalian conference of 2008, I was back in Amsterdam and excitedly emailed Ivan, asking if he had any idea how the photoelectron asymmetry was affected by the absorption of multi-photons instead of a single photon. Within a couple of hours, I received a very nice and comprehensive email about his work with single photon PECD and the fact that multi-photon PECD had not yet been demonstrated experimentally. His reply encouraged me greatly.

In our new lab in Amsterdam, we had built a powerful new electron-ion coincidence detector, so we could measure the electron angular scattering distribution in coincidence with the mass of the molecule on the opposing ion detector. So, for every molecule ionized in our set-up (even with different chiral molecules present simultaneously in mixtures), we could obtain the mass via time-of-flight, and the chirality by the coincident PECD of the electron angular distribution on the second detector. And all of this could be done with a commercial ultrafast laser. Suddenly, a trip to the synchrotron appeared to be no longer necessary – detection of enantiomers in chiral mixtures might be possible with our own tabletop instrument. And the idea of MS-PECD was born!

In reality, it took some time before we really started doing multiphoton chiral PECD experiments. But when a new postdoc from the renowned Tata Institute for Fundamental Research in Mumbai, India, arrived in my lab in 2010 – Bhargava Ram Niraghatam – and I began working with Stefan Lehmann, we were able to conduct our first laser-based chirality experiments. Bhargava and Stefan were a wonderful team and made rapid progress. We quickly got multi-photon PECD data on camphor; our tabletop MS-PECD technique worked! We took more time in the lab to improve the quality of the data and we worked hard on the proper analysis of our coincidence data. The multi-photon PECD effect that we measured was large, 8 percent forward-backward asymmetry in the photoionization of camphor at 400 nm.

We submitted our first MS-PECD results to Nature in September 2011, but the paper got rejected rather quickly. Looking back, this rejection was a blessing in disguise as it guided me to contact Ivan again. We needed a better theoretical understanding of multi-photon PECD. I invited Ivan to join us in our laser-based multi-photon PECD project to help us in understanding our results. Ivan accepted enthusiastically and from the fall of 2011, the laser-based MS-PECD project became a wonderful collaboration with Ivan. We published our results on camphor with solid data analysis, theoretical calculations and interpretation in the Journal of Chemical Physics (1) and were very pleased to receive the 2013 JCP Editor Award for groundbreaking research in chemical physics. In fact, we had successfully developed novel technology for tabletop, laser-based multi-photon MS-PECD detection of chiral molecules. If I do say it myself, our solution worked like a charm!

In early 2014, Ivan and I published an invited Perspective paper for Physical Chemistry Chemical Physics that reviewed both synchrotron-based single photon and laser-based multi-photon PECD (2). In it, we outlined the analytical potential of tabletop MS-PECD, especially in combination with the rapid advancements in ultrafast laser technology.

Experimentally, we wanted to demonstrate the potential of MS-PECD in a more analytical application, using various multi-component mixtures of chiral molecules. We wanted to measure the enantiomeric excess (ee) of the various molecules in the mixtures, without doing any prior enantiomeric preparations like chiral GC/LC enantiomeric separation or selective chiral complexation (the Kinetic ‘Graham Cooks method’). With a new graduate student from Iran in the lab – Mohammad Rafiee Fanood – we decided to make mixtures of limonene and camphor, and prepared mixtures with different ee.

The MS-PECD results turned out great. Of each chiral molecule in the mixture, we obtained the mass from the time-of-flight on the ion detector and the ee from the PECD asymmetry on the coincident electron detector. In June 2015, our results of the ee measurements in multi-component mixtures were published in Nature Communications (3). On the day that our paper was published, my new company – MassSpecpecD BV – was incorporated in Enschede, the Netherlands.

It was actually back in the spring of 2015 when I first took the leap of faith, leaving academia and transitioning to the world of technology transfer, innovation and (start-up) businesses to establish MassSpecpecD BV. We are now based on the campus of the entrepreneurial University of Twente.

Beyond the Solution

The mission of MassSpecpecD is simple: to drive MS-PECD technology into the laboratory-based analytical instrumentation market. We want to provide businesses and researchers who work with chiral molecules with an innovation that can help solve their questions and problems, aid research and development, and improve product quality control. Indeed, we believe that laser-based MS-PECD has unique advantages, capabilities and potential for end users.

The 20th century was the century of the electron. Discovered by J. J. Thomson in 1897, the electron literally electrified society and led to the relatively rapid integration of the transistor, integrated circuits, chips and computers into our lives. Electrical discharges and electron impact ionization have been part of MS technology since its employment in the first mass spectrometers by A. J. Dempster and F. W. Aston in around 1918. We are already some 15 years into the 21st century and for many it will be the century of the photon. When T. Maiman developed the first laser in 1960, some commented that it was a solution looking for a problem.

Fifty years later, it is clear that the laser ‘solution’ has found its problems. Photons, optics and lasers are rapidly proliferating in our (global) society, solving everyday problems, increasing human productivity, providing real-time communication around the world and opening novel applications that few even dreamt of. However, lasers and photons have found only limited employment in modern mass spectrometers. In fact, lasers have only been used in MALDI systems where few of the special features of photons and lasers are actually used; the tunability, frequency selectivity, short pulse duration, high fluence and high-repetition rate of modern lasers have all been largely ignored, so far, in commercial mass spectrometers – as has the potential for controlled soft-ionization coupled with the facile polarization control of photons. Until now.

The year 2015 was proclaimed by the United Nations as the International Year of Light and Light-based Technologies (www.light2015.org). MassSpecpecD BV was founded during that special year and we are very much looking forward to bringing the unique features and potential of photons and lasers as a novel solution in the analytical world of (chiral) mass spectrometry. In December of the Year of Light, our technology was recognized in The Analytical Scientist Innovation Awards (TASIAs). A good end to a 
good year!

Maurice Janssen is founder and CEO at MassSpecpecD BV, the Netherlands (www.massspecpecd.com)